Common Phenotypes and Solutions - Protein
Short Run Time -- Stops running at 35 seconds or less
Most Probable Causes
- Upper marker in ladder was not correctly assigned due to low intensity of upper marker in the ladder -- To correct for wrong selected upper marker in ladder, set upper marker manually; if necessary, adjust peak find settings; if peaks are detected that are not part of the ladder, exclude them; for better upper marker identification, turn of the analysis; for correct alignment, overlay electropherograms of multiple wells to clearly identify the upper marker
Additional Sample or Ladder Peaks
Most Probable Causes
- Sample or ladder not denatured properly -- Use fresh sample aliquot; head sample/denaturing solution and ladder for 5 min at 100C
- Sample/denaturing solution and/or ladder are dried out during denaturation -- Sample/denaturing solution and/or ladder were denatured in 1.5ml tubes; use 0.5ml tubes for denaturing
- Chip contaminated -- Wear powder-free gloves only; don't touch the wells of the chip
- Dust particles in separation channels -- Clean the electrodes; load the chip immediately after taking it out of its sealed bag
Probable Causes
- Ladder degraded -- Refer to the protein reagent kit guide for proper ladder storage; optional: prepare ladder aliquots and use a new aliquot
- Vibration of the bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from bench
Low or Missing Upper Marker in Ladder
Most Probable Causes
- Ladder degraded -- For correct ladder storage and denaturation, refer to the protein reagent kit guide; to correct for wrong selected upper marker, set upper marker manually; if necessary, adjust peak find settings; if peaks are detected that are not part of the ladder, exclude them; for better marker identification: turn off the analysis; for correct alignment, overlay elctropherograms of multiple wells to clearly identify the upper marker
- Diluted ladder is too old -- Use diluted ladder within one day
Probable Causes
- Ladder not denatured properly -- Use fresh ladder aliquot; heat ladder for 5 min at 100C
- Ladder dried out during denaturation -- Ladder was denatured in 1.5ml tubes; use 0.5ml tubes for denaturing
Low or Missing Upper Marker in Sample
Most Probable Causes
- Sample buffer/denaturing solution not handled according to the instructions -- Refer to the instructions provided with the reagent kit guide for storage and preparation of the sample buffer/denaturing solution; to correct for wrong selected upper marker, set upper marker manually; if necessary, adjust peak find settings; for better upper marker identification: turn off the analysis; for correct alignment, overlay electropherograms of multiple wells to clearly identify the upper marker
- Incompatible sample components that may interfere with the upper marker and decrease sensitivity -- See protein reagent kit guides for a list of compatible buffers and buffer compounds; if necessary, dilute, dialyze or desalt the sample; it is recommended to dilute the sample 1:2, 1:4, ... with water to find the optimal dilution
- Diluted samples are too old -- Use diluted samples within one day
Probable Causes
- Upper marker was digested by proteases -- Add protease inhibitor cocktails to cell lysate samples
- Samples not denatured properly -- Use fresh sammple aliquot; heat sample with denaturing solution for 5 min at 100C
- Samples dried out during denaturation -- Samples were denatured in 1.5ml tubes; use 0.5ml tubes for denaturing
High Lower Marker Variability -- Assay performance not affected by lower marker or system peak variability
Most Probable Causes
- Buffer components of the sample, e.g. salts, detergents, other additives etc. interfere with the lower marker -- See protein reagent kit guide for a list of compatible buffers and buffer compounds
- Variability of ionic strength of the sample influence the lower marker intensity -- If necessary, dilute, dialyze or desalt the sample; it is recommended to dilute the samples 1:2, 1:4,... with water to find the optimal dilution
Missing Peaks -- Few or no peaks observed
Most Probable Causes
- Laser defective -- Check laser using the Diagnostics test
- Gel dye mix was loaded in the destain well instead of destaining solution -- Discard chip and prepare new chip according to protocol
Probable Causes
- Autofocus failure -- Check autofocus using the Diagnostics test
Least Probable Causes
- High voltage power supply defective -- Check high voltage power supply using the Diagnostics test
- Fingerprint on focusing lens or on the backside of the chip -- Clean lens; do not touch the underside of the chip
Spikes -- Sharp up then down peaks
Most Probable Causes
- Chip/gel-dye mix/destaining solution/electrodes contaminated -- Prepare new chip with new gel-dye mix and new destaining solution; wear powder-free gloves only; don't touch the underside of the chip; don't touch the wells of the chip; clean the electrodes; load the chip immediately after taking it out of its sealed bag
- Gel-dye mix/destaining solution not properly prepared -- Refer to the reagent kit guide for proper preparation of the gel-dye mix and destaining solution
Probable Causes
- Vibration of the bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from bench
Poor Reproducibility
Most Probable Causes
- Wrong peak alignment -- Check if alignment is correct; for better identification of the lower and upper marker: turn off the analysis; for correct alignment, overlay electropherograms of multiple wells to clearly identify the lower and upper marker
- One or more samples not denatured -- Use fresh sample aliquot; heat samples with denaturing solution for 5 min at 100C
- One or more samples dried out during denaturation -- Samples were denatured in 1.5 ml tubes; use 0.5 ml tubes for denaturing
- Reducing agent was added in one sample and not in the other -- Refer to the reagent kit guide for proper sample reduction
- Dirty electrodes -- Thoroughly clean the electrodes
Probable Causes
- Diluted samples are too old -- Use diluted sample within one day
- Incompatible buffer component -- See protein reagent kit guide for a list of compatible buffers and buffer compounds; if necessary, dilute, dialyze or desalt the sample
Low Sensitivity -- Only minor peaks detected
Most Probable Causes
- Protein concentration in samples too low -- Use protein concentration according to specifications given in the reagent kit guide
- Too high salt concentration in samples -- Sensitivity is strongly affected by salt concentration; dilute samples in deionized water, dialyze samples against low salt buffer or desalt samples using spin filters
- SDS not completely dissolved in dye concentrate -- Let dye concentrate equilibrate to room temperature for 30 min before use; protect dye concentrate from light during this time; check for undissolved SDS crystals in the tube; vortex dye concentrate well before use; if necessary, heat the sample buffer to 37C for 2 min
- Samples were not diluted prior to chip loading -- Dilute samples according to protocol given in the reagent kit guide
Probable Causes
- Samples not completely denatured -- Use fresh sample aliquot; heat sample/denaturing solution for 5 min at 100C
- Sample/denaturing solution are dried out -- Sample/denaturing solution were denatured in 1.5 ml tubes; use 0.5 ml tubes for denaturing
- Pipetting error during preparation of mixtures -- Check dilution procedure and check calibration of pipette
Least Probable Causes
- Samples dissolved in acidic buffer -- Neutralize samples with appropriate buffer or dilute samples in deionized water; alternatively, dialyze samples against buffer with medium pH
Low Ladder Peaks -- Ladder peaks observed too soon
Most Probable Causes
Ladder degraded -- Refer to the protein reagent kit guide for proper ladder storage; optional: prepare ladder aliquots and use a new
aliquot
- Ladder not diluted after denaturation -- Refer to the Reagent Kit Guide for proper chip preparation
Probable Causes
- Ladder not completely denatured -- Use fresh ladder aliquot; head ladder for 5 min at 100C
- Ladder dried out -- Ladder was denatured in 1.5 ml tubes; use 0.5 ml tubes for denaturing
- Diluted ladder is too old -- Use diluted ladder within one day
- Pipetting error during preparation of mixtures -- Check dilution procedure and calibration of pipette
Broad Peaks -- Peaks are too wide
Most Probable Causes
- Wrong peak alignment -- Check if alignment is correct (wrong alignment might cause broad peaks compared to the rest of the chip); for better identification of the lower and upper marker: turn off the analysis; for correct alignment, overlay electropherograms of multiple wells to clearly identify the lower and upper marker
- Air bubbles in the bottom of the well -- Always insert the pipette tip to the bottom of the well when dispensing the liquid; remove large air bubbles with a pipette tip (small bubbles on top of well do not affect the assay)
- Chip not properly primed; clogged chip priming station or wrong settings of priming station -- Prepare a new chip; check chip priming station using the seal test; check if clip and base plate of priming station are in the correct position
- Leak current due to contaminated electrodes; chip was left in instrument after run -- Clean electrodes with analysis-grade water and a toothbrush; don't leave chip in instrument after run; clean electrodes after each run
Probable Causes
- Sample was not denatured properly -- Use fresh sample aliquot; heat sample/denaturing solution for 5 min at 100C
- Reducing agent was added in one sample and not in the other -- Refer to the reagent kit guide for proper sample reduction
Baseline Dips -- Assay performance not affected by dips as long as lower marker is detected
Most Probable Causes
- Sample contains additional detergents and/or dyes -- See protein reagent kit guide for a list of compatible buffers and buffer compounds; if necessary, dilute, dialyze or desalt the sample
Late Migration -- Peaks observed to be wider and later in run
Most Probable Causes
- Protein chip expired -- Check expiration date on chip box
- Protein concentration in samples too high -- Use protein concentration according to specifications given in the reagent kit guide
- Chip not properly primed; clogged chip priming station or wrong settings of priming station -- Prepare a new chip; check chip priming station using seal test; clean/replace gasket and plastic adapter if necessary; check if clip and base plate of priming station are in the correct position
Least Probable Causes
- Defective heater plate -- Run the temperature test
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